Characterization of dengue virus in Aedes aegypti and Aedes albopictus spp. of mosquitoes: A study in Khyber Pakhtunkhwa, Pakistan

Dengue is a vector-borne disease caused by dengue virus. According to the recent report of CDC that one-third population of the world are at high risk with Dengue fever. The prevalence of the dengue hemorrhagic fever was found more in tropical and sub-tropical regions of the world. Aedes mosquitoes was reported as the main cause of transmission of dengue virus. So the current study was planned to characterize the virus in Aedes mosquitoes collected from different area of Pakistan. In current investigation, Aedes mosquitoes and larvae were trapped under conducive conditions which are counted as 495 Aedes mosquitoes and 260 Aedes larvae. First of all, adult mosquitoes were identified morphologically under microscopy, counted as 73.3% Ae. aegypti and 26.7% Ae. albopictus. Finally, reverse transcriptase polymerase chain reaction analyses that only 4 adults of Aedes mosquitoes and 10 Aedes larvae as naturally infected with dengue virus with possible source Ae. aegypti. This study basically uncovered the presence of virus in different species of mosquitoes in southern regions of Pakistan. The present study will also give us an insight for vector control programs of dengue virus in the affected area.


INTRODUCTION
With more than one-third of the world's population living in areas at risk for infection, dengue virus is a leading cause of illness and death in the tropics and subtropics. As many as 400 million people are infected yearly. Dengue fever is Vector born disease and caused by any one of four related viruses transmitted by mosquitoes (CDC). Mosquitoes are medically and economically significant group of insects among dipterans that serves as vector for a various number of human and zoonotic disease pathogens [1]. Aedes aegypti is a competent vector of

MATERIALS AND METHODS
The present study was conducted in different areas of district Kohat latitude of 33,5869 (3335'12.840"N) and at a longitude of 71,4414 (7126'29.040"E) with an altitude of 489 meters (1,604 feet), Khyber Pakhtunkhwa from the month of June 2015 to June 2016. In district Kohat, during the month of August in summer and December in winter, maximum humidity (67%) has been reported. Aedes mosquitoes were trapped from different rural and urban areas of the study district before sunset and after sunrise, using Centers for Disease Control and Prevention (CDC) light traps. CDC traps were hanged at the height of about 6 feet from earth surface in places near plants, trees or containers having stagnant water.
The collected mosquitoes were placed in 70% ethanol. Different pots (containers, jars, bottles etc.) were also kept under observation for Aedes mosquito eggs, larvae, pupae, and adults. Collected adults and larvae of Aedes mosquitoes were placed in 70% ethanol brought to Vector-borne Disease Management center at Kohat University of Science and Technology (KUST), Khyber Pakhtunkhwa, Pakistan. Adults of Ae. Aegypti and Ae. albopictus were separated from other mosquitoes by their morphological characteristics under microscope.
RNA from adults and larvae of Aedes mosquitoes were extracted by Favor PrepTM Viral Nucleic Acid Extraction Kit I according to manufacturer instructions. RNA was stored at -20 °C for further use. The same protocol was repeated for Aedes larvae. RNA extracted from trapped Aedes mosquitoes and larvae, was converted to cDNA by using reverse transcriptase enzyme. cDNA was then pooled (cDNA of 20 mosquitoes per pool) separately for adults and larvae, MBRC http://mbrc.shirazu.ac.ir 79 reactions were performed. Reverse transcriptase enzyme and dengue virus reverse primer D2 were used to convert target sequence of the virus RNA into the complimentary copy of DNA (cDNA). After that forward dengue virus primer, D1 was used for amplification of cDNA. In 13 μl of RT mixture, 12 μl of extracted RNA was added. ٥×RT buffer was consisted of Tris-HCl =260 mM, pH = 8.4, KCl = 370 mM, MgCl2 = 20 mM, dNTP's = 1.3mM of each, D2 primer = 30 pmol, Superscript II= 250 units, RNase inhibitor = 20 units. This mixture was incubated at 37°C for 60 minutes and cDNA was synthesized. The enzyme was inactivated at 100°C for 10 minutes. Further, 50 μl volume was used for the first round of PCR. In 38 μl of PCR mixture, 12 μl of cDNA were added.
PCR was then performed in a 50 mL reaction volume using a set of five primers comprising a dengue virus consensus reverse primer and four serotype specific forward primers. The reaction contained of initial denaturation at 94°C for 5 minutes followed by 35 cycles (denaturation at 94°C for 1 minute, annealing at 55°C for 90 sec and extension at 72°C for 2.

RESULTS AND DISCUSSION
A total of 1150 mosquitoes were trapped from different locations of district Kohat by using CDC traps. Among them, 495 were Aedes mosquitoes. The highest number of mosquitoes was trapped during the month of August (data not shown); heavy rainfall occurs that causes the mosquitoes to grow in stagnant water. Aedes mosquitoes were trapped from different containers where water can be naturally stored including pots (jars, bottles, pitchers etc), unused buckets, drums, tyres, tree holes and cemented water reservoirs. These containers were already placed near patient's houses, poultry farms, dairy farms, and in open-aired areas. Previous studies reported the same results [13][14][15][16][17].
For the molecular study, RT-PCR of these pools was performed for amplification of dengue virus genome. RT-PCR results were positive for only four pools of adult Aedes mosquito samples (Table 1). And also for Aedes larvae samples, four pools show positive results and remaining pool's results were negative (Table 2).   Tyres act as a source of transport of dengue vectors (Aedes mosquitoes) not only within the country but also in different parts of the world because Aedes mosquitoes may breed in tyres if store in the water [18] and according to our study, tyres may be a factor of the outbreak in Lachi. In district, Kohat people mostly store water in different containers so Aedes mosquitoes easily breed there. That's why different areas of district Kohat, mosquitoes are increasing in number and can also disperse to other areas also is in close agreement with [19]. We point out that collected Aedes mosquitoes were identified as 61% female Aedes mosquitoes and 39% male Aedes mosquitoes of both species (Ae. aegypti and Ae. albopictus) which indicated that female Aedes mosquitoes were in higher number than male mosquitoes. This may be due to their blood feeding behavior from humans to complete life cycle. Similar observations have been observed by [20].